Ribosome-Mediated Incorporation of Dipeptides and Dipeptide Analogues into Proteins in Vitro

Article abstract of DOI:10.1021/jacs.5b03135

Plasmids containing 23S rRNA randomized at positions 2057-2063 and 2502-2507 were introduced into Escherichia coli, affording a library of clones which produced modified ribosomes in addition to the pre-existing wild-type ribosomes. These clones were screened with a derivative of puromycin, a natural product which acts as an analogue of the 3′-end of aminoacyl-tRNA and terminates protein synthesis by accepting the growing polypeptide chain, thereby killing bacterial cells. The puromycin derivative in this study contained the dipeptide p-methoxyphenylalanylglycine, implying the ability of the modified ribosomes in clones sensitive to this puromycin analogue to recognize dipeptides. Several clones inhibited by the puromycin derivative were used to make S-30 preparations, and some of these were shown to support the incorporation of dipeptides into proteins. The four incorporated species included two dipeptides (Gly-Phe (2) and Phe-Gly (3)), as well as a thiolated dipeptide analogue (4) and a fluorescent oxazole (5) having amine and carboxyl groups approximately the same distance apart as in a normal dipeptide. A protein containing both thiolated dipeptide 4 and a 7-methoxycoumarin fluorophore was found to undergo fluorescence quenching. Introduction of the oxazole fluorophore 5 into dihydrofolate reductase or green fluorescent protein resulted in quite strong enhancement of its fluorescence emission, and the basis for this enhancement was studied. The aggregate results demonstrate the feasibility of incorporating dipeptides as a single ribosomal event, and illustrate the lack of recognition of the central peptide bond in the dipeptide, potentially enabling the incorporation of a broad variety of structural analogues.

Full text of DOI:10.1021/jacs.5b03135

Communication
pubs.acs.org/JACS
Ribosome-Mediated Incorporation of Dipeptides and Dipeptide
Analogues into Proteins in Vitro
Rumit Maini, Larisa M. Dedkova, Rakesh Paul, Manikandadas M. Madathil, Sandipan Roy Chowdhury,
Shengxi Chen, and Sidney M. Hecht*
Center for BioEnergetics, Biodesign Institute, and Department of Chemistry & Biochemistry, Arizona State University, Tempe,
Arizona 85287, United States
S
* Supporting Information
ABSTRACT: Plasmids containing 23S rRNA randomized
at positions 2057−2063 and 2502−2507 were introduced
into Escherichia coli, affording a library of clones which
produced modified ribosomes in addition to the pre-
existing wild-type ribosomes. These clones were screened
with a derivative of puromycin, a natural product which
acts as an analogue of the 3′-end of aminoacyl-tRNA and
terminates protein synthesis by accepting the growing
polypeptide chain, thereby killing bacterial cells. The
puromycin derivative in this study contained the dipeptide
p-methoxyphenylalanylglycine, implying the ability of the
modified ribosomes in clones sensitive to this puromycin
analogue to recognize dipeptides. Several clones inhibited
by the puromycin derivative were used to make S-30
preparations, and some of these were shown to support
the incorporation of dipeptides into proteins. The four
incorporated species included two dipeptides (Gly-Phe (2)
and Phe-Gly (3)), as well as a thiolated dipeptide analogue
(4) and a fluorescent oxazole (5) having amine and
carboxyl groups approximately the same distance apart as
in a normal dipeptide. A protein containing both thiolated
dipeptide 4 and a 7-methoxycoumarin fluorophore was
found to undergo fluorescence quenching. Introduction of
the oxazole fluorophore 5 into dihydrofolate reductase or
green fluorescent protein resulted in quite strong enhance-
Figure 1. Puromycin derivative 1 used for selection of modified
ment of its fluorescence emission, and the basis for this
ribosomes, and dipeptides (2 and 3) and dipeptide analogues (4 and
enhancement was studied. The aggregate results demon-
5) incorporated into proteins using the modified ribosomes.
strate the feasibility of incorporating dipeptides as a single
ribosomal event, and illustrate the lack of recognition of
the central peptide bond in the dipeptide, potentially
enabling the incorporation of a broad variety of structural
analogues.
rRNA have been altered. Initial efforts involved the successful
incorporation of D-amino acids4 and β-amino acids.5 The
strategy employed for the incorporation of β-amino acids was
of special interest, involving the use of a puromycin analogue
containing a β-amino acid to enable the selection of promising
n recent years, many laboratories have employed ribosome-
Imediated protein synthesis to produce proteins containing
non-proteinogenic amino acids, enabling a more detailed study
of protein structure, function, and dynamics.1 While a broad
range of amino acids can now be incorporated into proteins,
the incorporation of α-amino acids containing large and
negatively charged side chains is still challenging,2 as is protein
biosynthesis with non α-amino acids.3
ribosomal architectures at the level of bacterial cells harboring
the plasmids with the modified 23S rRNAs.5a This study argued
that it may well be possible to mediate the incorporation of
other types of amino acids using ribosomes selected by the use
of puromycin analogues containing the amino acid structures of
interest. Presently, we employ a puromycin derivative (1)
containing the dipeptide p-methoxyphenylalanylglycine, and
demonstrate that the selected ribosomes successfully incorpo-
Recently, we have described the facilitated incorporation of
non-proteinogenic amino acids by the use of modified
ribosomes in which key regions of the Escherichia coli 23S
Received: March 25, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/jacs.5b03135
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Journal of the American Chemical Society
Communication
rated three dipeptides (2−4, Figure 1), including one dipeptide
having a thioamide linkage (4), as well as a fluorescent
dipeptidomimetic analogue (5, Scheme S1) reminiscent of the
fluorophores in natural fluorescent proteins.6
incorporation of 2 was accomplished by MALDI-MS analysis of
a tryptic digest of the elaborated DHFR (Figure S2) which
contained an ion at m/z 1377, corresponding to the peptide
fragment MISLIAALAGFDR, while DHFR containing phenyl-
alanine at position 10 had the analogous ion at m/z 1320,
corresponding to MISLIAALAFDR. A complete analysis of
fragment ions is summarized in Table S1. It may be noted that,
apart from clone 010326R6, the S-30 preparations derived from
an additional three of the nine unique clones identified could
also incorporate 2 and 3 into DHFR with comparable
suppression efficiency (not shown). Further, the DHFR
prepared from clone 010326R6 containing 2 at position 10
(Figure 2) was ∼84% as active as authentic wild-type DHFR,
providing a measure of the fidelity with which this S-30 system
was able to incorporate α-amino acids.9 The lower fidelity of
the modified ribosomes is unsurprising, and will likely prove to
be general for such ribosomes.
The bacterial clones employed for screening were part of a
previously described library containing modifications in specific
regions of 23S rRNA known to be involved in peptide bond
formation.5a,7 These were screened for inhibition by
dipeptidylpuromycin 1 (Figure 1), the preparation of which
is outlined in Scheme S2. A total of 419 clones were screened,
and 13 were inhibited by at least 50% in the presence of 100
μg/mL puromycin derivative 1. Nine of these clones,
containing the sequence 2057UGCGUGG2063 in their 23S
rRNA to confer erythromycin resistance, had also been
randomized in the region 2502−2507, and are summarized in
Table 1. As shown, four of these nine clones proved to be
Thiopeptide moieties have been shown to be useful as
fluorescence quenchers in peptides and proteins, functioning
Table 1. Sequence in Region 2502−2507 of Clones Having
the Same Sequeuce (UGCGUGG) in Region 2057−2063
both by Forster resonance energy transfer (FRET)10 and by
̈
photoinduced electron transfer (PET).11 This property has
been utilized very effectively by the Petersson laboratory for
characterization of protein structure following introduction of
the requisite thioamide by native chemical ligation of a
synthetic peptide fragment containing the thioamide to the
remainder of the ribosomally produced protein.10b,12,13 Since
the modified ribosomes described here could plausibly provide
an alternative route to proteins containing thioamides at
predetermined positions, we incorporated a thiolated dipeptide
(4) into position 16 of DHFR and the fluorescent probe L-(7-
methoxycoumarinyl-4-yl)ethylglycine, which is known to be
sensitive to environment,14 into position 49.15
The requisite thiolated dipeptide was obtained as outlined in
Scheme S4. Fully protected phenylalanylglycine was converted
to the respective thiodipeptide (24) by treatment with
Lawesson’s reagent.16 Following conversion to the N-pentenoyl
protected cyanomethyl ester (26), condensation with pdCpA
afforded the thiolated dipeptidyl-pdCpA ester, the latter of
which was converted to thiophenylalanylglycyl-tRNACCCG via
the agency of T4 RNA ligase. In analogy with an earlier study,17
thiophenylalanylglycyl-tRNACCCG was used to introduce 4 into
position 16 of DHFR, and (7-methoxycoumarin-4-yl)-
ethylglycyl-tRNACUA was used to introduce the fluorophore
into position 49. Another modified DHFR was prepared
containing phenylalanylglycine at position 16 and the 7-
methoxycoumarin fluorophore at position 49. As shown in
Figure 3A, when equimolar amounts18 of the two samples were
excited at 310 nm, only the DHFR containing thiophenyl-
alanine at position 16 underwent fluorescence quenching.19
While the mechanism was not studied in detail, given the
distances involved (Figure 3B)15 it seems likely that this
occurred by photoinduced electron transfer.
identical, having the sequence 2502ACGAAG2507, and
another two shared the sequence 2502CUACAG2507. Clone
010326R6 was chosen for further study based on its inhibition
by 1 and the favorable properties of the S-30 system prepared
from this clone in cell-free protein synthesis experiments.
As shown in Scheme S3, glycylphenylalanine (2) was
protected as its N-pentenoyl derivative and used to esterify
the dinucleotide pdCpA.8 Bacteriophage T4 RNA ligase was
then employed to ligate the dinucleotide to an in vitro RNA
transcript, affording glycylphenylalanyl-tRNACUA. Phenylalanyl-
glycine (3) was used to prepare phenylalanylglycyl-tRNACUA
analogously (not shown). When utilized in an S-30 system
prepared from clone 010326R6, and in the presence of E. coli
dihydrofolate reductase (DHFR) mRNA containing a UAG
codon corresponding to position 10 of DHFR, the dipeptidyl-
tRNACUAs effected suppression of the UAG codon, producing
DHFRs containing 2 (8% suppression) and 3 (14%
suppression) at position 10 (Figure 2). Verification of the
In addition to the foregoing three dipeptides, dipeptido-
mimetic analogue 5 was also investigated. This compound lacks
the peptide bond which connects the amino acids in dipeptides
2−4, but has a similar distance between the amine and
carboxylate groups which participate in incorporation of the
dipeptide into the protein backbone. Oxazole 5 is fluorescent,
having λex at 302 nm, and λem at 403 nm in water.20 It was
prepared and used to activate tRNACUA as outlined in Scheme
S1. The incorporation of this oxazole within DHFR at position
10 was verified by MALDI-MS of a tryptic digest (Figure S5).21
Incorporation by the S-30 preparations from two different
Figure 2. Autoradiogram of a SDS−polyacrylamide gel showing the
translation of DHFR from wild-type (lane 1) and modified (lanes 2−
4) (UAG codon in position 10) mRNA in the presence of different
suppressor tRNACUAs using an S-30 system prepared from clone
010326R6. Lane 2, non-acylated tRNACUA; lane 3, glycylphenylalanyl-
tRNACUA; lane 4, phenylalanylglycyl-tRNACUA. The suppression
efficiency relative to wild type is shown below each lane. Additional
incorporation data is provided in Figure S1.
B
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Journal of the American Chemical Society
Communication
Figure 4. Fluorescence emission spectra of (blue trace) free oxazole 5
and (red trace) oxazole 5 present at position 10 of DHFR (both were
present at 100 nM concentration in 25 mM Tris-HCl, pH 7.4,
containing 0.5 M NaCl; excitation at 302 nm). The spectrum of
oxazole 5 was obtained on the protected oxazole (12) to mimic the
form of the oxazole present within full length DHFR. The emission
spectrum of oxazole 12 was also recorded at higher and lower
concentrations (Figure S6).
Figure 3. (A) Fluorescence emission of two samples of DHFR (10 ng/
μL), each having 7-methoxycoumarin in position 49 and Phe-Gly (3)
(blue trace) or thioPhe-Gly (4) (orange trace) in position 16 after
excitation at 310 nm in 25 mM Tris-HCl, pH 7.4, containing 0.5 M
NaCl. (B) Model of the DHFR construct containing the 7-
methoxycoumarin fluorophore in position 49 and thio-Phe-Gly (4)
at position 16, based on DHFR structure PDB 1RA1. The distance
from the oxygen of Met16 (corresponding to the S atom of ThioFG in
the figure, yellow color) to the α-carbon of Ser49 is 7.7 Å.
Figure 5. Fluorescence emission spectra of (orange trace) green
fluorescent protein containing oxazole 5 at position 66 (excitation at
310 nm); (red trace) green fluorescent protein containing oxazole 5 at
position 39 (excitation at 310 nm); (blue trace) blue fluorescent
protein (excitation at 375 nm); (green trace) green fluorescent protein
(excitation at 395 nm), all present at 2.5 ng/μL concentration in 25
mM Tris-HCl, pH 7.4, containing 0.5 M NaCl.
Table 2. Incorporation of Oxazole 5 into Position 10 of E.
coli DHFR by the Use of S-30 Systems Having Different
Modified Ribosomes
fluorescent protein (BFP, chosen for comparison due to its
shorter emission wavelength than GFP).
suppression efficiency in different
sequence in 23S rRNA
of modified ribosomes
S-30 systems having modified
ribosomes (%)
Since the BFP fluorophore is known to be greatly enhanced
by its inclusion within the β-barrel structure, this suggests that
the inclusion of the oxazole fluorophore within the protein
backbone alone is sufficient to increase its fluorescence signal
significantly. When the oxazole was introduced into position 66
of GFP in lieu of Tyr66 (GFP66oxazole5), the resulting GFP
derivative had fluorescence emission several-fold stronger than
BFP or GFP, and had its emission shifted to shorter
wavelength, undoubtedly due to its presence within the β-
barrel structure. It may be noted that, unlike the typical
fluorophores formed in the natural and modified fluorescent
proteins, the oxazole fluorophore is completely stable, and
requires no preactivation to exhibit fluorescence.
The absorption extinction coefficients and quantum yields of
the GFP analogues containing 5 were determined in
comparison with that of wild type GFP (Table 3). The GFP
analogues had absorption extinction coefficients and quantum
yields greater than those of wild-type GFP, and entirely
consistent with the fluorescence emission data shown in Figure
5.
region 1
region 2
amino acid
(2057−2063)
(2502−2507)
−
5
UGCGUGG
AGUGAGA
ACGAAG
AUCCGA
0.8 0.2
2.2 1.0
15.3 2.5
14.0 3.0
clones was achieved with 14−15% suppression efficiency
(Table 2). It was also found to result in a dramatic increase
in the intensity of fluorescence emission and a shift in the
emission maximum to ∼395 nm, reflecting its sensitivity to its
environment (Figure 4). We posit that the inclusion of the
oxazole fluorophore within the protein backbone may be
sufficient to increase its fluorescence signal significantly. To test
this hypothesis, we incorporated the same oxazole at two
positions of green fluorescent protein (GFP) (Figure 5).
In one construct, GFP was rendered nonfluorescent by
mutating Tyr66 (which is essential for fluorophore generation)
to glycine, and then introducing the oxazole into position 39 of
GFP (GFP66Gly39oxazole5), which is outside (but close to)
the β-barrel structure in which the GFP fluorophore normally
resides.6 This species emitted at 410 nm (Figure 5) with an
intensity 4−5-fold greater than that of wild-type blue
As illustrated by the foregoing examples, we anticipate that it
will be possible to incorporate a variety of dipeptide analogues
into proteins and other biomaterials. The lack of participation
of the central amide bond of the dipeptide in the process by
C
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Journal of the American Chemical Society
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Chowdhury, S.; Alcala-Torano, R.; Hecht, S. M. Bioorg. Med. Chem.
2013, 21, 1088−1096. (c) Maini, R.; Roy Chowdhury, S.; Dedkova, L.
M.; Roy, B.; Daskalova, S. M.; Paul, R.; Chen, S.; Hecht, S. M.
Biochemistry 2015, 54, 3694−3706.
Table 3. Estimation of Quantum Yields of Modified Green
Fluorescent Proteins in Comparison with GFPwt
λex/abs, abs extinction coeff,
protein
nm
M−1 cm−1
quantum yield, λem
(6) Tsien, R. Y. Annu. Rev. Biochem. 1998, 67, 509−544.
GFPwt
395
310
310
25,000
90,300
50,200
0.79 (509 nm)22
0.91 (378 nm)
0.84 (407 nm)
(7) Modifications to the 23S rRNA also included one of eight new
sequences for nucleotides 2057−2063, introduced to confer moderate
resistance to erythromycin in support of the selection protocol.5a
(8) Robertson, S. A.; Noren, C. J.; Anthony-Cahill, S. J.; Griffith, M.
C.; Schultz, P. G. Nucleic Acids Res. 1989, 17, 9649−9660.
(9) While the S-30 preparation contained both modified and wild-
type ribosomes, only the modified ribosomes can incorporate the
dipeptide, and so must be the source of all DHFR polypeptide chains
containing the dipeptide. Position 10 of DHFR has no known
function,2a so incorporation of Gly-Phe at position 10 per se would not
be expected to alter DHFR activity. See Table S2 for complete data.
(10) (a) Goldberg, J. M.; Batjargal, S.; Petersson, E. J. J. Am. Chem.
Soc. 2010, 132, 14718−14720. (b) Wissner, R. F.; Batjargal, S.; Fadzen,
C. M.; Petersson, E. J. J. Am. Chem. Soc. 2013, 135, 6529−6540.
(11) (a) Goldberg, J. M.; Speight, L. C.; Fegley, M. W.; Petersson, E.
J. J. Am. Chem. Soc. 2012, 134, 6088−6091. (b) Goldberg, J. M.;
Batjargal, S.; Chen, B. S.; Petersson, E. J. J. Am. Chem. Soc. 2013, 135,
18651−18658.
GFP66oxazole5
GFP66Gly39oxazole5
which the dipeptide is incorporated implies that it is essentially
expendable structurally, as illustrated by analogues 4 and 5.
Thus, the incorporation of dipeptide mimetics can afford
structures not accessible by the successive incorporation of two
amino acids. Numerous applications, including the creation of
libraries of fluorescent proteins having a range of photophysical
properties23 and the metabolic stabilization of proteins of
therapeutic interest,24 can be readily envisioned. While
structure 5, in particular, represents a fairly significant departure
from any amino acid whose ribosomal incorporation into
protein has been reported previously, it may ultimately prove
feasible to select ribosomes capable of incorporating even more
complex and potentially useful substrates.
(12) Batjargal, S.; Wang, Y. J.; Goldberg, J. M.; Wissner, R. F.;
Petersson, E. J. J. Am. Chem. Soc. 2012, 134, 9172−9182.
(13) Muir, T. W. Annu. Rev. Biochem. 2003, 72, 249−289.
(14) Murakami, H.; Hohsaka, T.; Ashizuka, Y.; Hashimoto, K.;
Sisido, M. Biomacromolecules 2000, 1, 118−125. (Figures 7−9 in this
report employ three amino acids containing the 7-methoxycoumarin
fluorophore, each of which was attached to the backbone of
streptavidin at position 120 via one of three different linkers. Their
fluorescence emission maxima differed by about 20 nm, establishing
the environmental sensitivity of this fluorophore.)
(15) In an earlier study two pyrenylalanines incorporated into these
positions were within several angstroms, and close enough to undergo
excimer formation. See: Chen, S.; Wang, L.; Fahmi, N. E.; Benkovic, S.
J.; Hecht, S. M. J. Am. Chem. Soc. 2012, 134, 18883−18885 and
references therein.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.5b03135.
■
S
Synthetic methods, compound characterization for
compounds prepared, and additional characterization of
the elaborated proteins (PDF)
AUTHOR INFORMATION
Corresponding Author
*sidhecht@asu.edu
Notes
■
(16) Thomsen, I.; Clausen, K.; Scheibye, S.; Lawesson, S.-O. Org.
Synth. 1984, 62, 158−164.
The authors declare no competing financial interest.
(17) Chen, S.; Fahmi, N. E.; Wang, L.; Bhattacharya, C.; Benkovic, S.
J.; Hecht, S. M. J. Am. Chem. Soc. 2013, 135, 12924−12927.
(18) Quantification of protein concentrations was carried out by
Coomassie Brilliant Blue R-250 staining of bands of the proteins
following separation by SDS−PAGE, in comparison with known
concentrations of wild-type DHFR (Figure S3).
ACKNOWLEDGMENTS
■
This work was supported by National Institutes of Health
Research Grant GM103861, awarded by the National Institute
of General Medical Sciences. We thank Dr. Sriloy Dey for
assistance in preparing a synthetic intermediate.
(19) Also prepared in parallel was a modified DHFR containing
thiodipeptide 4 at position 10. Analysis of a tryptic digest by MALDI-
MS verified incorporation of the thiolated dipeptide, as well as the
absence of any detectable exchange of O in lieu of S (Figure S4).
(20) For oxazole derivative 12, the molar absorptivity in MeOH was
26 800 M−1 cm−1 and the quantum yield was 0.59.
(21) To date, we have been unable to verify dipeptide incorporation
by MS/MS analysis, relying instead on MS data derived from
proteolytic fragments. We note that these do not reflect any increase in
microheterogeneity occasioned by the use of modified ribosomes.
(22) Patterson, G. H.; Knobel, S. M.; Sharif, W. D.; Kain, S. R.;
Piston, D. W. Biophys. J. 1997, 73, 2782−2790.
(23) We have recently succeeded in incorporating a fluorescent
thiazole amino acid into GFP. Roy Chowdhury et al., submitted.
(24) Kamionka, M. Curr. Pharm. Biotechnol. 2011, 12, 268−274.
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